1. Field of the Invention
The present invention in the fields of immunology and virology relates human monoclonal antibodies specific for the CD4-binding domain of HIV gp120 and their uses in neutralizing HIV and in treating HIV infection. Also provided is a composition comprising a mixture of at least two antibodies specific to different HIV gp120 epitopes, preferably in the V3 domain and the CD4-binding domain, which synergize in the neutralization of HIV.
2. Description of the Background Art
The human immunodeficiency virus (HIV) has been implicated as the causative agent of acquired immune deficiency syndrome (AIDS). Two different HIV families have been identified to date: HIV-1 and HIV-2. It is currently believed that the majority of individuals that become infected with HIV eventually will develop AIDS and are likely to succumb to fatal infections and/or malignancies. Currently, it is estimated that approximately 1.5 million persons have been infected by HIV in the United States alone. Thus, treatment and prevention of HIV infection is among the leading public health challenges today.
Chemotherapy of patients afflicted with AIDS or HIV infections with antiviral drugs, such as azidothymidine (AZT), has led to some clinical and immunological improvement and has decreased the mortality rate and frequency of opportunistic infections. However, such drugs are expensive, may induce resistant strains of HIV, and have numerous toxic side effects, and may therefore not be suitable for administration to all AIDS patients.
Immunotherapeutic approaches to AIDS include use of monoclonal antibodies (mAbs) of defined specificity directed against HIV-1 proteins expressed in infected patients. These HIV-1 virion proteins, expressed by infected cells, are designated inter alia as p24, gp41, gp120, etc. (See, for example, Essex, U.S. Pat. No. 4,725,6569.
Passive immunization has been used to prevent and treat many bacterial and viral illnesses (Zolla-Pazner S. et al., J. Virol. Meth. 17:45-53 (1987)) and has been shown to prevent the infection of chimpanzees with HIV-1 (Emini E. A. et al., J. Virol. 64:3674-3678 (1990); Prince A. M. et al., In: Vaccines 90: Modern Approaches to New Vaccines Including Prevention of AIDS, Brown, F., ed, Cold Spring Harbor Laboratories Press, Cold Spring Harbor, N.Y., 1990, pp. 347-351). Given these precedents, it is likely that passive immunotherapy may be useful in (a) preventing HIV infection in health care workers after accidental exposure, (b) blocking transmission of HIV from the infected mother to her fetus and (c) treating previously HIV-infected patients. It is generally thought that useful antibodies for the treatment of HIV infection are those capable of neutralizing HIV, by either simple binding to a virion component or by antibody-mediated inhibition of viral functions such as binding to the cell surface receptor, the CD4 molecule. Furthermore, it is generally accepted that human mAbs to HIV would be preferable because of their lack of foreign antigenic sites that may lead to a host immune response against the antibody. An optimal passive immunotherapeutic agent would comprise a human antibody preparation which could be administered intramuscularly and which would neutralize a majority, if not all, HIV-1 strains.
A number of human monoclonal antibodies (mAbs) to HIV have been produced. Several of the human mAbs specific for gp120 possess neutralizing activity (Gorny M. K. et al., Proc. Natl. Acad. Sci. (USA) 88:3238-3242 (1991); Robinson J. E. et al., AIDS Res. Hum. Retrovir. 6:567-579 (1990); Posner M. R. et al., J. Immunol. 146:4325-4331 (1991); Ho D. D. et al., J. Virol. 65:489-493 (1991); Tilley, S. A. et al., Research Virol. 142:247-259 (1991)). These mAbs have generally been categorized as "type-specific" or "group-specific". Type-specific mAbs are generally directed against the V3 loop of gp120 (Goudsmit J. et al., FASEB J. 5:2427-2436 (1991); Nara P. L. et al., FASEB J. 5:2437-2455 (1991)) and by definition are restricted in their biologic function to one or a few related HIV-1 isolates. Some group-specific mabs are directed against a large conformational region of gp120 responsible for binding to CD4 (Goudsmit et al., supra; Nara et al., supra); these mAbs generally neutralize many (but not all) HIV-1 isolates.
Some of the neutralizing epitopes of HIV are located in conserved regions of the proteins (Ho, D. D. et al., Science 239: 1021 (1988)). However, they do not seem to elicit particularly strong immune responses nor to serve as appropriate neutralizing domains since (a) serum titers of antibodies recognizing these epitopes are generally low, and (b) high concentrations of murine mAbs to these epitopes are required to achieve neutralization.
Several studies have reported that the principal neutralizing epitope of HIV-1 appears to reside in the V3 domain of gp120 (Goudsmit, J. et al., Proc. Natl. Acad. Sci. USA 85:4478-4482 (1988); Goudsmit, J. AIDS 2:S41 (1988); Matsushita, S. M. et al., J. Virol. 62:2107 (1988); Javaherian, K. et al., Proc. Natl. Acad. Sci. USA 86:6768 (1989); Kowalski et al., Science 237:1351 (1987); Palker et al., Proc. Natl. Acad. Sci. USA 85:1758-1762 (1988)). The V3 loop has a "tip" which consists of an essentially conserved sequence of four amino acids, Gly-Pro-Gly-Arg (G-P-G-R; SEQ ID NO:1) flanked by amino acid residues which vary among HIV-1 isolates. The V3 loop has been shown to be important for infectivity (Stephens et al., Nature 343:219 (1990); Hattori et al., FEBS Lett. 248:48 (1989)).
The interaction of the gp120 external envelope glycoprotein with CD4 is the principal step leading to the entry of HIV-1 into CD4+ cells (Dalgleish, A. G. et al., Nature 312:763-767 (1984)). The structure of the CD4-binding domain of gp120 is not yet fully elucidated. Substitution of individual amino acids, deletion of several residues, or truncation of large C-terminal fragments altered CD4-gp120 binding (Cordonnier, A. et al., Nature 340:571-574 (1989); Kowalski, M. et al., Science 237:1351-1355 (1987); Ardman, B. et al., J. AIDS 3:206-214 (1990)). Olshevsky et al. (J. Virol. 64:5701-5707 (1990)) recently demonstrated that structure of the CD4-binding domain included contributions from the second, third, and fourth conserved regions of gp120, thus indicating that it is a conformational epitope.
In natural infections, the immune responses to the V3 loop and the CD4-binding domain are quite different. While antibodies to the V3 loop develop early in the course of HIV infection, their titer appears to wane during the first year or two (Blattner, W. et al., Int'l. Conf. AIDS 5:510 (1989)). In contrast, antibodies to the CD4-binding domain arise more slowly but appear to persist for longer periods (Ho D. D. et al., J. Virol. 65:489-493 (1991)). Since a CD4-binding domain is common to all competent HIV-1 viruses, antibodies to this region, which are generally neutralizing, would be expected to offer a relatively broad range of protection. Thus, in order to (a) better characterize the human immune response to HIV, (b) formulate vaccines that can induce broadly protective antibodies and (c) use such antibodies in passive immunotherapy, it is important to understand the specificity of these antibodies and the mechanism by which they protect against disease.
Monoclonal antibodies (mAbs) to the CD4-binding domain of gp120 have previously been disclosed. Some of these inhibit CD4-gp120 binding but do not neutralize virus infectivity (Sun, H. et al., J. Virol. 63:3579-3585 (1989)). Other mAbs, both murine (Sun, H. et al., supra) and human (Robinson J. E. et al., supra; Posner M. R. et al., supra; Tilley, S. A. et al., supra), inhibit CD4-gp120 binding and also neutralize HIV infectivity. At least three neutralizing mAbs (two murine and one human) specific for the CD4-binding domain do not cross-compete (Ho, D. D. et al., J. Virol. 65:489-493 (1991)). These findings, in combination with the current structural understanding of the CD4-binding domain, provide further support for the conclusion that the CD4-binding domain exists as a large, conformation-dependent, discontinuous epitope on the surface of the gp120 molecule.
Thus, for purposes of both research and therapy, there is a recognized need in the art for human mAbs with broad virus group specificity to the CD4-binding domain. In particular, it would be advantageous to obtain such mAbs using a variety of screening techniques to insure the diversity of the mAbs selected. The production of new human mAbs specific for the CD4-binding domain of gp120 thus constitute one objective of the present invention (see below).
Finally, synergy between antibodies has been demonstrated in the neutralization of a number of viruses (Della-Porta A. J. et al., J. Gen. Virol. 38:1-19 (1977); Henchal E. A. et al. J. Gen. Virol. 69:2101-2107 (1988); Wensvoort G., J. Gen. Virol. 70:2865-2876 (1989)). Studies from the present inventors' laboratory showed that different human mAbs specific for the HIV glycoprotein gp41 acted synergistically. However the effect of this synergy was not better neutralization but rather antibody-dependent enhancement of HIV infection (Robinson, W. E. Jr. et al., Proc. Natl. Acad. Sci. (USA) 87:3185-3189 (1990); Robinson W. E. Jr et al., J. Virol. 65:4169-4176 (1991)).
Tilley et al. (Proc. 7th Int'l. Conf. on AIDS, Florence, Italy (Jun. 16-21, 1991), abstr. M.A.70) disclosed that a V3 specific human mAb, 4117C, acted in concert with a CD4-binding domain-specific human mAb, 1125H, in neutralizing HIV. Importantly, however, the V3 antibody had a limited range of reactivity, reacting with V3 of MN, SF-2 and NY-5 isolates of HIV but not with RF or IIIB strains. Furthermore, this reference did not determine whether the two antibodies were synergistic.
It is therefore important to identify synergistic interactions between other, more broadly reactive, anti-HIV antibodies that result in both improved virus neutralization and a broader range of reactivity, which can be exploited in AIDS immunotherapy.